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l-Lactic Acid Production via Sustainable Neutralizer-Free Route by Engineering Acid-Tolerant Yeast .Journal of Agricultural and Food... Jul 2023l-Lactic acid (l-LA) is a platform chemical obtained via microbial fermentation at a near-neutral pH value. Large amounts of neutralizers are required during this...
l-Lactic acid (l-LA) is a platform chemical obtained via microbial fermentation at a near-neutral pH value. Large amounts of neutralizers are required during this process, which increases the production costs in downstream processing as well as environmental burden. To address this challenge, an acid-tolerant yeast E1 was isolated and metabolically engineered to produce l-LA without neutralizers. The genome of strain E1 was sequenced and a CRISPR-Cas9 system was developed in this newly isolated strain. Subsequently, the gene encoding pyruvate decarboxylase () was knocked out to subdue ethanol formation. Furthermore, the l-lactate dehydrogenase gene from 2-6 and the codon-optimized gene from were introduced into E1 chromosome to redirect the ethanol fermentation pathway to l-LA production. Deletion of the () gene further increased the optical purity of l-LA. After optimizing fermentation conditions, the maximum titer of l-LA in the 5 L fermenter reached 74.57 g/L without any neutralizers, with an optical purity of 100% and a maximum yield of 0.93 g/g glucose. This is the first report of optically pure l-LA production without neutralizers and the engineered acid-tolerant yeast paves the way for the sustainable production of l-LA via a green route.
Topics: Animals; Cattle; Saccharomyces cerevisiae; Lactic Acid; Acids; Pichia; Fermentation; Ethanol
PubMed: 37439413
DOI: 10.1021/acs.jafc.3c03163 -
Applied Microbiology and Biotechnology Aug 2023Saccharomyces cerevisiae is the workhorse of fermentation industry. Upon engineering for D-lactate production by a series of gene deletions, this yeast had deficiencies...
Saccharomyces cerevisiae is the workhorse of fermentation industry. Upon engineering for D-lactate production by a series of gene deletions, this yeast had deficiencies in cell growth and D-lactate production at high substrate concentrations. Complex nutrients or high cell density were thus required to support growth and D-lactate production with a potential to increase medium and process cost of industrial-scale D-lactate production. As an alternative microbial biocatalyst, a Crabtree-negative and thermotolerant yeast Kluyveromyces marxianus was engineered in this study to produce high titer and yield of D-lactate at a lower pH without growth defects. Only pyruvate decarboxylase 1 (PDC1) gene was replaced by a codon-optimized bacterial D-lactate dehydrogenase (ldhA). Ethanol, glycerol, or acetic acid was not produced by the resulting strain, KMΔpdc1::ldhA. Aeration rate at 1.5 vvm and culture pH 5.0 at 30 °C provided the highest D-lactate titer of 42.97 ± 0.48 g/L from glucose. Yield and productivity of D-lactate, and glucose-consumption rate were 0.85 ± 0.01 g/g, 0.90 ± 0.01 g/(L·h), and 1.06 ± 0.00 g/(L·h), respectively. Surprisingly, D-lactate titer, productivity, and glucose-consumption rate of 52.29 ± 0.68 g/L, 1.38 ± 0.05 g/(L·h), and 1.22 ± 0.00 g/(L·h), respectively, were higher at 42 °C compared to 30 °C. Sugarcane molasses, a low-value carbon, led to the highest D-lactate titer and yield of 66.26 ± 0.81 g/L and 0.91 ± 0.01 g/g, respectively, in a medium without additional nutrients. This study is a pioneer work of engineering K. marxianus to produce D-lactate at the yield approaching theoretical maximum using simple batch process. Our results support the potential of an engineered K. marxianus for D-lactate production on an industrial scale. KEY POINTS: • K. marxianus was engineered by deleting PDC1 and expressing codon-optimized D-ldhA. • The strain allowed high D-lactate titer and yield under pH ranging from 3.5 to 5.0. • The strain produced 66 g/L D-lactate at 30 °C from molasses without any additional nutrients.
Topics: Lactic Acid; Saccharomyces cerevisiae; Kluyveromyces; L-Lactate Dehydrogenase; Glucose; Pyruvate Decarboxylase; Hydrogen-Ion Concentration; Fermentation
PubMed: 37405435
DOI: 10.1007/s00253-023-12658-2 -
Plant Physiology and Biochemistry : PPB Aug 2023Hypoxic stress due to submergence is a serious threat to the growth and development of maize. WRKY transcription factors are significant regulators of plant responses to...
Hypoxic stress due to submergence is a serious threat to the growth and development of maize. WRKY transcription factors are significant regulators of plant responses to various abiotic and biotic stresses. Nevertheless, their function and regulatory mechanisms in the resistance of maize to submergence stress remain unclear. Here we report the cloning of a maize WRKY transcription factor gene, ZmWRKY70, transcripts of which accumulate under submergence stress in maize seedlings. Subcellular localization analysis and yeast transcriptional activation assay indicated that ZmWRKY70 was localized in the nucleus and had transcriptional activation activity. Heterologous overexpression of ZmWRKY70 in Arabidopsis increased the tolerance of seeds and seedlings to submergence stress by upregulating the transcripts of several key genes involved in anaerobic respiration, such as group VII ethylene-responsive factor (ERFVII) (AtRAP2.2), alcohol dehydrogenase (AtADH1), pyruvate decarboxylase (AtPDC1/2), and sucrose synthase (AtSUS4), under submergence conditions. Moreover, the overexpression of ZmWRKY70 in maize mesophyll protoplasts enhanced the expression of ZmERFVII members (ZmERF148, ZmERF179, and ZmERF193), ZmADH1, ZmPDC2/3, and ZmSUS1. Yeast one-hybrid and dual-luciferase activity assays further confirmed that ZmWRKY70 enhanced the expression of ZmERF148 by binding to the W box motif located in the promoter region of ZmERF148. Together, these results indicate that ZmWRKY70 plays a significant role in tolerance of submergence stress. This work provides a theoretical basis, and suggests excellent genes, for biotechnological breeding to improve the tolerance of maize to submergence through the regulation of ZmWRKY genes.
Topics: Arabidopsis; Zea mays; Saccharomyces cerevisiae; Plant Breeding; Transcription Factors; Stress, Physiological; Seedlings; Gene Expression Regulation, Plant; Plants, Genetically Modified; Plant Proteins
PubMed: 37364509
DOI: 10.1016/j.plaphy.2023.107861 -
Plant Physiology Sep 2023Root growth in maize (Zea mays L.) is regulated by the activity of the quiescent center (QC) stem cells located within the root apical meristem. Here, we show that...
Root growth in maize (Zea mays L.) is regulated by the activity of the quiescent center (QC) stem cells located within the root apical meristem. Here, we show that despite being highly hypoxic under normal oxygen tension, QC stem cells are vulnerable to hypoxic stress, which causes their degradation with subsequent inhibition of root growth. Under low oxygen, QC stem cells became depleted of starch and soluble sugars and exhibited reliance on glycolytic fermentation with the impairment of the TCA cycle through the depressed activity of several enzymes, including pyruvate dehydrogenase (PDH). This finding suggests that carbohydrate delivery from the shoot might be insufficient to meet the metabolic demand of QC stem cells during stress. Some metabolic changes characteristic of the hypoxic response in mature root cells were not observed in the QC. Hypoxia-responsive genes, such as PYRUVATE DECARBOXYLASE (PDC) and ALCOHOL DEHYDROGENASE (ADH), were not activated in response to hypoxia, despite an increase in ADH activity. Increases in phosphoenolpyruvate (PEP) with little change in steady-state levels of succinate were also atypical responses to low-oxygen tensions. Overexpression of PHYTOGLOBIN 1 (ZmPgb1.1) preserved the functionality of the QC stem cells during stress. The QC stem cell preservation was underpinned by extensive metabolic rewiring centered around activation of the TCA cycle and retention of carbohydrate storage products, denoting a more efficient energy production and diminished demand for carbohydrates under conditions where nutrient transport may be limiting. Overall, this study provides an overview of metabolic responses occurring in plant stem cells during oxygen deficiency.
Topics: Plant Roots; Oxygen; Meristem; Stem Cells; Hypoxia; Carbohydrates
PubMed: 37311198
DOI: 10.1093/plphys/kiad344